descriptionGenomic losses of the 9p21.3 region encompassing the CDKN2A/2B genes are frequent events in many human cancers. This locus encodes three cyclin-dependent kinase inhibitors p14ARF, p16INK4A and p15INK4B (see schematic presentation below). Genomic deletion of one or both copies of these important cell cycle regulator genes is the main inactivation mechanism. CDKN2A deletion can extend to the MTAP gene, located 110 kb away. The MTAP gene encodes methylthioadenosine phosphorylase, an important enzyme for the salvage of both adenine and methionine. It is known that many tumour cells require addition of methionine to their growth medium, because their MTAP gene is co-deleted with CDKN2A. Cells lacking MTAP are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation, and therefore homozygous co-deletion of the CDKN2A and MTAP genes might open possibilities for alternative treatment for cancer patients. Other genes that are frequently co-deleted with CDKN2A/2B are MIR31, CDKN2B-AS1 and PAX5. Loss of MIR31 has been shown to have pro-tumorigenic effects on e.g. breast and ovarian cancer (Creighton CJ et al. 2010, Cancer Res. 70:1906-15). CDKN2B-AS1 (non-protein coding CDKN2B antisense RNA 1) is suggested to act as an epigenetic silencer of the CDKN2B gene (Yu W et al. 2008, Nature. 451:202-6). The PAX5 gene, at 9p13, which is essential for normal B-cell lymphopoiesis, is frequently co-deleted with CDKN2A in B-ALL (Kim M et al. 2011, Blood Cells Mol Dis. 47:62-6).

An alternative mechanism of inactivation of the CDKN2A/2B genes is the hypermethylation of the promoter regions leading to lack of expression of p14, p15 and p16 proteins, which further results in uncontrolled cell proliferation and tumour development and progression.

Alterations of the CDKN2A/2B genes are described not only in somatic tumour samples but also in the germline. Germline mutations in the CDKN2A gene have been linked to development of malignant cutaneous melanoma in some families with hereditary melanoma. Up to 40% of familial melanoma predisposition cases are associated with CDKN2A mutations (Hewitt C et al. 2002, Hum Mol Genet. 11:1273-9). Next to point mutations, various intragenic deletions within this gene have been identified in hereditary melanoma. The SALSA® MLPA® P419 Familial melanoma probemix contains more probes for CDKN2A/2B, and probes for CDK4. This P419 probemix can be used both on germline DNA and on tumour DNA.

This ME024-B2 MS-MLPA probemix contains 23 probes for the CDKN2A/2B gene region (including two probes for CDKN2B-AS1), ten of which are MS-MLPA probes which detect the methylation status of the promoter regions of these tumour suppressor genes. The MIR31, MTAP and PAX5 genes are covered by two probes each. Four more probes target the region between the MIR31 gene and the 9p telomere. Besides detecting aberrant methylation, all 33 target probes present will give information on copy number changes in the analysed sample. Additionally, 12 reference probes, not affected by HhaI digestion and located on stable genomic regions in most tumour types, and two digestion control probes that can be used to confirm complete digestion by the HhaI enzyme are included.

The MS-MLPA probes in this ME024-B2 probemix detect sequences in promoter regions of tumour suppressor genes that are unmethylated in most blood-derived DNA samples. Upon digestion, the peak signal obtained in unmethylated samples will be very small or absent. In contrast, when tested on in vitro methylated human DNA, these probes do generate a signal. We have no data showing that methylation detected by a particular probe indeed influences the corresponding mRNA levels.

Schematic presentation of CDKN2A and CDKN2B gene structure and proteins encoded by these genes

Note: An alternative exon 4 (330 nt before exon 4, present in NG_007485.1 sequence) was published in March 2011 and is present in NM_001195132.1 transcript variant 5. Clinical significance of this transcript variant is not yet known. This variant is also known as p16ɣ (Lin YC et al. 2007, Oncogene. 26:7017-27). Transcript variant 5 includes an additional exon that causes a frameshift in the 3' coding region when compared to variant 1 (encoding p16INK4A). The resulting isoform p16ɣ has a distinct C-terminus, which is longer than p16INK4A, and this isoform is a candidate for nonsense-mediated mRNA decay. However, it is not known if this endogenous protein p16ɣ is expressed in vivo.

This SALSA® MS-MLPA® probemix can be used to detect aberrant methylation of one or more sequences of promoter regions of the CDKN2A and CDKN2B genes. Methylation levels can be different for different tissues. If possible, use identically treated test and reference samples (same tissue type and extraction method). This SALSA® MS-MLPA® probemix can also be used to detect copy number changes (deletions/duplications) of one or more sequences in the above mentioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA® MS-MLPA® test. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons.

product historyversion B2: One flanking probe has been redesigned, two reference probes have been replaced and several probes have a small change in length but no change in the sequence detected.version B1: One additional probe for CDKN2A has been included and several probes for genes MIR31, CDKN2B-AS and PAX5 on 9p arm have been included. Several reference probes have been replaced/included. 88 and 96nt control fragments have been replaced (QDX2).version A1: As compared to P024: The number of CDKN2A and CDKN2B probes have been increased from 13 to 20 and extra control fragments at 88-960-100-105 nt have been added. This kit can also be used for methylation detection of the p14, p15 and p16 promotor regions.